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As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is
committed to providing our customers around the world with the highest quality products.
In addition to direct selling in more than 20 countries in North America, Europe and
Asia/Pacific, Miltenyi Biotec also provides support for our customers through an
extensive distributor network covering dozens of additional countries.

As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is
committed to providing our customers around the world with the highest quality products.
In addition to direct selling in more than 20 countries in North America, Europe and
Asia/Pacific, Miltenyi Biotec also provides support for our customers through an
extensive distributor network covering dozens of additional countries.

Application protocol

In this application protocol, we describe the depletion of murine lineage cells from bone marrow samples, viral transduction of the remaining bone marrow cells, and their phenotypic analysis using flow cytometry.

MACS Columns and MACS Separators: Choose the appropriate MACS Separator and MACS Columns according to the number of labeled cells and the number of total cells. Use autoMACS for automated magnetic cell separation. ▲ Note: Use LS Columns for the Direct Lineage Cell Depletion Kit, mouse. The kit can be additionally used with the MultiMACS™ Cell24 Separator Plus, and includes a completely automated cell isolation protocol for use with the autoMACS Pro.

▲ Note: When using the Direct Lineage Cell Depletion Kit, the unwanted cell fraction is labeled and the targetcells remain unlabeled. Depending on the target cell frequency, the labeled fraction can represent the majority of the total cells. To avoid blocking of the column, do not exceed the maximum number of labeled cells per column. Estimate the number of labeled cells in the sample, split the sample if necessary and use the appropriate number of separation columns.

▲ Note: If separating with LS Columns and the MultiMACS Cell24 Separator Plus, use the Single-Column Adapter. Refer to the user manual for details.

Deplete cells expressing lineage markers from total bone marrow mononuclear cells using either the Direct Lineage Cell Depletion Kit, mouse or the Lineage Cell Depletion Kit, mouse. The first generates a high cell yield. The latter enables a more stringent depletion. Follow the protocol of the corresponding kit data sheet.

▲ Note: We recommend filtering the magnetically labeled cells before separation to guarantee a single-cell suspension either manually or via the automated protocol using the autoMACS® Pro Cell Separator.

Direct Lineage Cell Depletion Kit, mouse

Before separation

After separation

LSK staining

Isolation of untouched lineage-negative cells from a mouse bone marrow cell suspension. After isolation with the DIrect Lineage Cell Depletion Kit, mouse, lineage-negative cells were fluorescently stained with Lineage Cell Detection Cocktail-Biotin and Anti-Biotin-APC and analyzed by flow cytometry using the MACSQuant® Analyzer. To evaluate the LSK (Lin–Sca-1+c-kit+) fraction, cells were further stained with CD117-PE (c-kit) and Anti-Sca-1-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Clear separation of lineage-negative and lineage-positive cells. Untouched lineage negative cells from a mouse bone marrow cell suspension using the Lineage Cell Depletion Kit and a MidiMACS™ Separator with an LS Column. Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.

Clear separation of lineage-negative and lineage-positive cells

A comparison of the ungated forward scatter versus side scatter profile of the same sample after separation with the CD34 MicroBead Kit or the CD34 MicroBead Kit UltraPure demonstrates reduced debris carry-over with the CD34 MicroBead Kit UltraPure.

Isolation of lineage–/CD117+ cells from a mouse bone marrow cell suspension. After depletion of lineage+ cells using the Lineage Cell Depletion Kit, CD117 MicroBeads and a MidiMACS Separator with LS Columns were used to isolate CD117+ cells. The cells were then fluorescently stained with CD117-PE, and a panel of biotinylated antibodies against lineage markers and Anti-Biotin-APC. Cell debris and dead cells were excluded from the analysis.

Transduce the lineage marker-negative cells with a retroviral or lentiviral vector, either directly after MACS® enrichment or after pre-stimulation culture in appropriate cell culture medium supplemented with cytokines such as SCF, TPO, FLT-3L and IL-3 and IL-6.

Use Vectofusion-1®, a novel transduction enhancer, to boost transduction efficiency. Follow the protocol of the data sheet.

Lineage marker-negative cells can be expanded in appropriate mouse HSC expansion media supplemented with cytokines such as SCF, TPO, FLT-3L and IL-3 and IL-6 according to standard cell culture protocols.